1. Field of the Invention
This invention is directed to an apparatus that can be used in methods of preparing, amplifying, detecting, and/or optionally selecting for further analysis the genomic material from an organism for the rapid detection and/or classification of an organism in a sample (e.g., screening for, identifying, quantifying, and/or optionally further analyzing, e.g., sequencing, the genomic material of the organism).
2. Related Background Art
Recent basic and innovative developments have allowed biotechnological processes to become more sophisticated and simultaneously more complicated. For example, although many useful techniques have been developed to reduce the cost of, simplify, and standardize processes of DNA preparation, amplification, detection, and identification, there are no known apparatuses on the market that allow the full automation of these processes for the screening, quantification, identification, and/or further analysis, e.g., sequencing, of DNA.
In the biotechnological field, there is a need for rapid detection and/or classification of organisms, such as bacteria and viruses, in a variety of samples (e.g., environmental and medical). For example, rapid detection of bacteria, and subsequent classification of the species and/or strain, may be necessary to provide quality assurance for, e.g., a local water supply, a hospital, or a food processing plant; i.e., it may be necessary to monitor various samples, including but not limited to samples of air, dust, water, blood, tissues, plants, foodstuffs, etc., for the presence of contaminating organisms, and to classify the contaminating organisms prior to consumption, exposure, and/or use by the public, or during use by the public.
Standard microbiological methods for detecting and/or classifying an organism, e.g., culturing and Gram-staining or testing of other biochemical properties, are imprecise and often cannot differentiate among different organisms, let alone different strains of an organism. More precise methods for detecting and/or identifying an organism are based on the genomic DNA of the organism. One such well-known method of detection and/or identification (classification) is the polymerase chain reaction (PCR), for which technological developments have increased its level of throughput and automation.
PCR is effectuated by two separate and distinct (first and second) primers, each of which is respectively complementary to a nucleotide sequence found on either of the two templates of the genomic DNA. Since the sequences of the two primers are based on the sequences of the two genomic DNA templates, the two primers bind to and bracket a singular and isolated locus of the double-stranded genomic DNA. PCR using such a pair of primers results in the exponential amplification of double-stranded genomic DNA that is identical to the singular and isolated locus of the genome bracketed by nucleotide sequences complementary to the two primers, i.e., a locus of DNA flanked by a first primer binding site on the 3′-end of one genomic DNA template and a second primer binding site on the 3′-end of the other genomic DNA template.
PCR is useful in detecting small amounts of DNA, not only because it results in the exponential amplification of double-stranded DNA, but also because of the development of new technologies that increase the level of PCR throughput and automation. An example of one such technology is the use of microfluidic systems, including controller/detector interfaces for microfluidic devices, as described in, e.g., U.S. Pat. Nos. 6,500,323 and 6,670,153. These microfluidic systems, collectively referred to herein as automated inline PCR platforms, are well known in the art and are generally described below.
Most automated inline PCR platforms utilize a microfluidic chip that works with controller/detector interfaces for automated sample accession, microfluidic PCR reagent assembly, PCR thermal cycling, and optical detection spectroscopy. A microfluidic chip generally comprises a first plate with at least one micro-etched fluidic (microfluidic) inline reaction channel that may be bonded to a second plate, within which may be metal traces and a fluid reservoir. When the two plates are bonded together to form the microfluidic chip, each microfluidic reaction channel of the first plate may connect with a fluid reservoir of the second plate so that locus-specific reagents can be delivered through the fluid reservoirs to the microfluidic inline reaction channels.
Usually, automated inline PCR using a microfluidic chip does not occur in a chamber; instead, the reaction occurs as the sample is moved along and inside a microfluidic inline reaction channel. Inline PCR begins when a capillary, or “sipper,” aspirates a sample droplet (which may or may not be a DNA sample droplet, i.e., a sample droplet comprising genomic material isolated from an organism) from, e.g., a microtiter plate (which may come from, e.g., a robotic handler) into at least one microfluidic inline reaction channel. After aspirating a sample droplet into a microfluidic inline reaction channel, the sipper can be moved to a buffer trough so that buffer is drawn into the microfluidic chip. Consequently, cross-contamination among sample droplets is minimized since each sample droplet is separated from adjacent sample droplets by buffer spacers. Each sample droplet is then moved along a microfluidic inline reaction channel and into a PCR assembly area of the chip, wherein the sample droplet becomes a sample plug by being mixed with PCR-required reagents, e.g., a primer pair, DNA polymerase, and dNTPs, and detectable agents, e.g., intercalators, etc. Optionally, buffer spacers may also be mixed with PCR-required reagents to serve as negative controls. After being mixed with PCR-required and detectable agents, a sample plug (which may or may not be a DNA sample plug, i.e., a sample plug comprising genomic material) is moved along the length of the microfluidic inline reaction channel into different areas of the chip, e.g., an amplification area wherein PCR may be effected on the sample plugs.
Generally, as each sample plug (e.g., a DNA sample plug) flows through a microfluidic inline reaction channel, it enters an amplification area, i.e., a temperature-controlled area, wherein each microfluidic inline reaction channel is repeatedly and rapidly heated and cooled in a localized manner such that the denaturing, annealing and elongation steps of PCR are effected on each sample plug as it moves through the channel. A skilled artisan will recognize that amplification of DNA will occur only in DNA sample plugs, i.e., sample plugs comprising genomic material. A method of controlling the temperature in the amplification area is Joule heating (see, e.g., U.S. Pat. Nos. 5,965,410 and 6,670,153). Generally, voltage can be applied to the metal traces in a controlled and localized manner to effectuate the different temperatures required for each cycle of PCR (i.e., each cycle of denaturing, annealing, and elongation). Cooling of the reaction can be achieved through the use of, e.g., cooling fluid that travels through a coil to carry away thermal energy in the form of heat from the microfluidic inline reaction channel, or by allowing rapid heat dissipation, e.g., via the application of cold water to the bottom surface of the microfluidic chip. Since the volume of fluid in the microfluidic channels is small and the metal traces are located very close to the microfluidic inline reaction channels, heating and cooling of the fluid in the channels (and hence, sample plugs) is accomplished very rapidly. Consequently, DNA sample plugs undergo PCR, and PCR cycles run such that, e.g., 30 cycles may be performed in less than nine minutes. The number of PCR cycles each DNA sample plug sees as it travels through a microfluidic channel in the temperature-controlled area of the chip may be varied by changing either or both 1) the timing of the voltage applied to the metal traces, and 2) the flow rate of the DNA sample plugs through the microfluidic channels.
A microfluidic chip can simultaneously perform as many polymerase chain reactions as it has microfluidic inline reaction channels. For example, a sample comprising genomic material may be aspirated into multiple different microfluidic inline reaction channels, to each of which is added a different locus-specific reagent (e.g., a different primer pair that brackets a different locus on the genomic material, e.g., DNA). This allows for the simultaneous detection of several different loci on, e.g., genomic material isolated from the same organism. Alternatively, reagents comprising one specific primer pair may be aspirated into multiple different microfluidic inline reaction channels. This allows for the simultaneous detection of the same locus, e.g., on genomic material isolated from different organisms. Additionally, multiple sample droplets may be aspirated into the same microfluidic reaction channel.
A detection area is usually downstream of the temperature-controlled amplification area, and is generally a transparent region that allows observation and detection of the amplified DNA products, e.g., PCR products. In the detection area, each microfluidic inline reaction channel is usually brought in close proximity and passed under a detector. A light source is spread across the microfluidic inline reaction channels so that detectable agents, e.g., fluorescence emitted from each channel, e.g., from each DNA sample plug, passing through the optical detection area may be measured simultaneously. After the detection area, each microfluidic inline reaction channel usually leads each sample plug to a waste well.
Three different methods are usually used to generate fluid motion within microfluidic inline reaction channels; the methods involve electrokinetics, pressure, or a hybrid of the two (see, e.g., U.S. Pat. No. 6,670,153). In a pressure-based flow system, an internal or external source may be used to drive the flow of fluid in the inline reaction channels. For example, a vacuum may be applied to waste wells at the ends of each microfluidic inline reaction channel and may be used to activate the sipper and move the fluid along the microfluidic inline reaction channels toward the waste wells. Alternatively, since genomic material is charged, electrokinetics, i.e., the generation of a voltage gradient (e.g., by the application of voltage to the metal traces) may be used to drive charged fluid along the microfluidic inline reaction channels. A third method of driving the fluid along the inline reaction channels uses both electrokinetics and pressure. The result is a continuous flow of fluid within the microfluidic inline reaction channels, wherein sample plugs (e.g., DNA sample plugs) are continuously being mixed or moved to different areas (e.g., a PCR assembly area, a temperature-controlled area, a detection area, etc.) of the chip.
Electrokinetic and/or pressure-driven fluid movement, heating and cooling cycles, detection, and the data acquisition related to a microfluidic chip may be controlled by an instrument that interfaces at or with the chip (generally described in, e.g., U.S. Pat. No. 6,582,576). The interface of the instrument usually contains o-ring seals that seal the reagent wells on the chip, pogo pins that may interface with the metal traces on the chip and supply the voltage for temperature cycling, o-ring seals for the waste wells where a vacuum may be applied to move the fluid through the chip, a large o-ring that may be used to seal the bottom of the chip against circulating cool water and to speed the cooling during the temperature cycling, and a detection zone for, e.g., fluorescence detection. A skilled artisan will recognize that the risk of contamination with this system is minimal because a microfluidic chip is usually a closed system, physical barriers (e.g., buffer spacers) separate sample plugs (e.g., DNA sample plugs), and the continuous flow prevents sample plugs from moving backwards.
Since PCR (and consequently, automated inline PCR platforms) exponentially amplifies DNA, it may be used to detect small amounts of genomic material. However, because PCR requires primers that are specifically complimentary to sequences of the genomic material that are known and bracket the locus of interest, it is limited in that it can only be used for the detection and classification of known organisms. In other words, the investigator is required to know or guess the identity of the organism (i.e., the appropriate pair of primers to use) prior to any attempts at detecting the organism. Another limitation of PCR (and consequently of automated inline PCR platforms) is the inability of the investigator to obtain sequence information about the amplified DNA, other than information about the sequences complimentary to the two primers used in the analysis. Additionally, an automated inline PCR platform does not provide a means to further analyze, e.g., sequence, the genomic material in, e.g., a DNA sample plug, after it has traveled the length of a microfluidic inline reaction channel. Further analysis, e.g., providing the sequence, of the genomic material may be important and useful in, e.g., distinguishing a pathogenic strain from a nonpathogenic strain, detecting and providing the sequence of a new strain, etc.
To overcome some of the limitations of PCR, methods of waveform profiling were developed (see, e.g., the method of waveform profiling described in Japanese Patent Application Publication Nos. 2003-334082 and 2003-180351). Waveform profiling methods, e.g., those described in Japanese Patent Application Publication Nos. 2003-334082 and 2003-180351, provide ways to analyze and profile genomic material, e.g., DNA isolated from organisms, such as bacteria, without requiring the investigator to know or guess the identity of the organism prior to detection. Briefly, waveform profiling generally analyzes the genomic DNA of the organism using a unique primer(s) and the two denatured strands of the genomic DNA as templates to linearly amplify several distinct single-stranded nucleic acid polymers that form higher-order structures, e.g., triplexes, tetraplexes (or quadruplexes), etc. Because the genomic DNA of the organism is used as the template, the resulting single-stranded nucleic acid polymers will be distinct and contain sequences unique to the organism. Thus, the single-stranded nucleic acid polymers will form higher-order structures based on sequences unique to the organism. Accordingly, detection of such unique higher-order structures, which may be accomplished using detectable agents, e.g., fluorescent intercalators, may identify the organism.
The several distinct single-stranded nucleic acid polymers are usually produced using a single pattern generative waveform primer characterized by its structure and length. A waveform primer (i.e., a waveform-profiling primer) generally consists of two portions, a nonspecific stabilizing portion and a specific portion. As discussed below, the nonspecific stabilizing portion may help guide the formation of higher-order structures. In contrast, the specific portion guides the waveform primer to specifically bind to sequences complementary to its own sequence. The length of the waveform primer (e.g., 8-30 bases in length) is usually critical because it allows the specific portion of the primer to bind specifically to several discrete primer binding sites, i.e., sequences complementary to the waveform primer, along the length of a genomic DNA template. The binding of waveform primers to several primer-binding sites along each single-stranded genomic DNA template allows for the generation of several distinct single-stranded nucleic acid polymers, the generation of which is usually critical to this method.
In addition to utilizing a waveform primer, this method of waveform profiling also utilizes several cycles of linear amplification to provide multiple copies of each of several distinct single-stranded nucleic acid polymers; therefore, many copies of the waveform primer are added to a solution containing the genomic DNA of interest prior to the first cycle of linear amplification. Similar (at least generally) to PCR, one cycle of linear amplification comprises the following steps: 1) denaturing each copy of the double-stranded genomic DNA into two single-stranded genomic DNA templates, 2) annealing (i.e., providing conditions that allow the binding of) the waveform primer to several discrete primer binding sites on each single-stranded genomic DNA template, and 3) elongating several distinct single-stranded nucleic acid polymers from each of several waveform primers bound to primer binding sites along each genomic DNA template.
During one cycle of linear amplification, the temperature of the genomic DNA is increased (e.g., to 95-98° C.) to denature each copy of the genomic DNA into two single-stranded genomic DNA templates. The temperature is subsequently decreased (e.g., to 25° C.) to allow waveform primers to bind to several discrete primer-binding sites along the length of each denatured genomic DNA template. The final step in the cycle, elongation of several distinct single-stranded nucleic acid polymers from each bound waveform primer, is performed at ˜72° C. using a polymerase, e.g., Taq polymerase. After this final step, the cycle repeats.
During the next denaturing step, the several distinct nucleic acid polymers are denatured from the genomic DNA templates and become single-stranded nucleic acid polymers, wherein each single-stranded nucleic acid polymer has a 5′-to-3′ nucleotide sequence comprising the nucleotide sequence of the waveform primer from which the single-stranded nucleic acid polymer was elongated, followed by a distinct nucleotide sequence that is complementary to the sequence of the region of the genomic DNA template that was downstream of the genomic DNA sequence that bound to a waveform primer. Since each single-stranded nucleic acid polymer comprises the sequence of the waveform primer at its 5′-end, each single-stranded nucleic acid polymer also comprises the nonspecific stabilizing portion of the waveform primer. The nonspecific stabilizing portion of the waveform primer generally guides each single-stranded nucleic acid polymer to form higher-order structures and effectively prevents the single-stranded nucleic acid polymers from binding to any waveform primer in subsequent cycles of amplification.
In other words, the single-stranded nucleic acid polymers are not used as templates in subsequent cycles of amplification, and each cycle of amplification in this method of waveform profiling is linear and not exponential, i.e., each cycle of amplification produces only a single copy of each of the several distinct single-stranded nucleic acid polymers containing sequences unique to the organism, i.e., sequences complementary to sequences of the genomic DNA template that are downstream of waveform primers bound to primer binding sites. Thus, in contrast to PCR, which results in exponential amplification, waveform-profiling methods generally result in linear amplification, i.e., nonexponential amplification, of the several distinct single-stranded nucleic acid polymers containing sequences unique to the organism.
Each single-stranded nucleic acid polymer contains a base sequence complementary to a sequence of a genomic DNA template that is downstream of a waveform primer bound to a primer-binding site, so differences in base sequences present on multiple sites of different genomic DNAs may be compared and distinguished. As described above, the multiple copies of each of several distinct single-stranded nucleic acid polymers will interact with each other to form higher-order structures, i.e., complexes (e.g., triplexes and tetraplexes) comprising one or more single-stranded distinct nucleic acid polymers. The higher-order nucleic acid structures will have different stabilities and dissociate at different melting temperatures (Tm) depending on the base sequences of single-stranded nucleic acid polymers, i.e., based on the unique genomic information of the organism.
Waveform profiling generally requires that the Tm of the various different higher-order structures, produced using the genomic DNA of a particular organism as a template, be determined and recorded (melting temperature analysis); this can be accomplished with the use of fluorescent agents that intercalate into higher-order DNA structures, i.e., intercalators. The higher-order DNA structures generated by waveform profiling may be dissociated by increasing the temperature of the sample. As the higher-order DNA structures dissociate, the fluorescent agents intercalated in these higher-order structures will also dissociate. Plotting the rate of change of fluorescence intensity obtained by the dissociation of these higher-order structures as a function of increasing temperature will produce a waveform that is unique to the genomic DNA of the organism and the utilized waveform primer, i.e., the dissociation of higher-order DNA structures at different melting temperatures (Tm) are observed and recorded to produce a characteristic “waveform profile” for each species (or strain) of organism, e.g., bacteria. Thus, waveform profiling may be used to distinguish between genomic DNA isolated from a first organism and genomic DNA isolated from a second organism using melting temperature analysis and intercalators to obtain a unique waveform profile for each organism.
Since the above-described method (related to waveform profiling) relies on linear amplification, one of the difficulties of using this method is the requirement for a large starting amount of genomic DNA from the particular organism (e.g., bacteria) to be detected and/or identified. Consequently, waveform-profiling methods may be used to detect and identify organisms only if the organisms are present in large numbers (e.g., 106 or more organisms) within a given sample, but are not effective for detecting and/or identifying a very small number of organisms. Additionally, similar to PCR, another limitation of this method is its inability to provide detailed information about the genomic material, e.g., sequence information.
Accordingly, waveform profiling methods are generally not useful in detecting and/or identifying an organism present in small numbers, e.g., in a sample taken from a water supply or source at the onset of contamination, or providing detailed information, e.g., sequence information, about the genomic material of the organism. Although PCR (and consequently, inline automated PCR platforms) may resolve the limitation of this waveform profiling method that requires a large starting sample (since PCR results in the exponential amplification of the genomic DNA and allows for the detection of organisms present in small numbers), it is known in the art that waveform profiles produced using the complementary double-stranded pieces of DNA that result from PCR amplification are insufficient for identification of particular genomic sequences (see, e.g., “Goodbye DNA Chip, Hello Genopattern for 21st Century,” printed and distributed by Adgene Co., Ltd.). Also, to date, there is no known automated inline PCR platform capable of detecting waveform profiles. In other words, the prior art not only explicitly teaches it is not possible to compare, differentiate and identify genomic material (from various species or strains of organisms) using melting temperature (Tm) analysis of standard PCR products, it also fails to provide technology that increases the levels of waveform profiling throughput and automation.
Additionally, although waveform profiling methods may provide for the rapid detection and/or classification of an organism via detection of its genomic DNA, these methods, as well as methods of PCR and inline automated PCR platforms, are all limited because they do not provide detailed information on the genomic material, e.g., sequence information, as provided by a sequencing chip (see, e.g., U.S. Published Patent Application No. 2005/0009022). Further examination of the genomic material, e.g., analysis of the sequence information, may be important, for example, when genomic variations among different strains of the same organism (which may be undetectable using, e.g., a particular PCR primer pair or waveform primer) cause the different strains to have different pathogenic properties, in the detection of new strains of infectious agents (e.g., variants of influenza virus or variants of a biological weapon), which may pose greater threats to public health, etc.
As described above, many basic methods (e.g., PCR, waveform profiling, etc.) and innovative technological developments (e.g., automated inline PCR platforms) have taken place in the field of detecting and/or classifying organisms. Although these methods and developments are becoming more sophisticated, and have simplified, standardized, and made more efficient the detection and/or classification of organisms, the present inventors know of no art-recognized apparatus that provides for the automation of all of these methods and developments simultaneously, i.e., an automated inline platform that allows for PCR, waveform profiling, and/or optionally selecting genomic material for further analysis, e.g., sequencing. The present invention overcomes this limitation by providing such an apparatus comprising microfluidic devices that may be used to detect and/or classify (e.g., screen for, quantify, identify, and/or optionally select for further analysis, e.g., sequencing of) genomic material (isolated from an organism (e.g., bacteria or viruses)) in a sample by automated methods of preparing (e.g., isolating, processing, mixing with reaction reagents, etc.), amplifying (e.g., by PCR, waveform profiling, etc.), detecting and/or optionally selecting for further analysis, e.g., sequencing.